Constituents of the Stem Bark of Trichilia monadelpha (Thonn.) J. J. De Wilde (Meliaceae) and Their Antibacterial and Antiplasmodial Activities

The chemical investigation of the EtOH extract from the stem bark of Trichilia monadelpha (Thonn.) J. J. De Wilde afforded two new limonoids (1 and 2): 24-acetoxy-21,25-dihydroxy-21,23-epoxytirucall-7-en-3-one (1) and (6R)-1-O-deacetylkhayanolide E (2), together with eleven known compounds (3–13), including additional limonoids, flavonoids, triterpenoids, steroids, and fatty acid. Their structures were determined using 1D- and 2D-NMR experiments, ESI mass spectrometry, and single crystal X-ray diffraction analysis. The antibacterial and antiplasmodial activities of the extracts, sub-extracts, fractions, and some of the isolated compounds were evaluated in known pathogenic strains, including Staphylococcus aureus and Plasmodium falciparum. Fraction E (n-Hex/EtOAc 30:70, v/v) showed significant activity against S. aureus ATCC 25923 with a MIC value of 3.90 µg/mL, while one of its constituents (epicatechin (9)) exhibited significant activity with MIC values of 7.80 µg/mL. Interestingly, grandifotane A (6) (IC50 = 1.37 µM) and khayanolide D (5) (IC50 = 1.68 µM) were highly active against the chloroquine-sensitive/sulfadoxine-resistant plasmodium falciparum 3D7 strain, unlike their corresponding plant extract and fractions.


Plant Material
The stem bark of Trichilia monadelpha was collected in Mawa in the Noun sub-division of the West Region in Cameroon in September 2016, and subsequently identified by comparing it to a voucher specimen (66909/HNC) at the National Herbarium of Cameroon (NHC), Yaoundé.

X-ray Crystallography
X-ray crystallography data of (6R)-1-O-deacetylkhayanolide E (2): A colorless crystal was obtained from CH 2 Cl 2 -MeOH (3:7). Cell parameters: orthorhombic, space group P2 1  Four Gram-negative bacteria (Salmonella typhi, Pseudomonas aeruginosa NR 48982, Klebsiella pneumoniae NR 41388, and Klebsiella pneumoniae clinical isolate) and three Grampositive bacteria (Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 43300, and Staphylococcus aureus clinical isolate) were tested for their susceptibility to extracts and compounds isolated from T. monadelpha. The minimum inhibitory concentration (MIC) of the samples was evaluated following the broth microdilution method as described by Eloff [25], with slight modifications. Extracts, fractions, sub-fractions, compounds, and the reference drug were dissolved in DMSO. The bacterial suspension prepared as an inoculum was adjusted to a turbidity equivalent to that of a 0.5 McFarland standard to achieve approximately 1.5 × 10 8 CFU/mL. Ciprofloxacin was used as a positive control. A volume of one hundred microliters of Mueller Hinton Broth (MBH) was added into all wells of the 96-well plate, and 100 µL of the compounds/extracts, fractions, and sub-fractions were introduced to the wells in the first row (A), and then mixed thoroughly. A volume of 100 µL of this sample mixture was removed from the wells of row A to perform a two-fold serial dilution down the rows (B-H). The last 100 µL was discarded. Then, 100 µL of the inoculum was introduced into all the wells. The final volume in each well was 200 µL. Each sample concentration was assayed in triplicate, and each test was performed twice. After an incubation period of 18 h at 37 • C, 20 µL of Alamar Blue was added to each well. The plates were then reincubated for 30 min at 37 • C. A blue color in the well was scored as "no bacterial growth", and a pink color was scored as "growth occurrence". MIC values were defined as those concentrations in which a pronounced change in color was noticed (from blue to pink).

In Vitro Antiplasmodial Activity: Plasmodium falciparum Culture and Growth Inhibition Assay
Plasmodium falciparum 3D7 (chloroquine-sensitive/sulfadoxine-resistant) strain was maintained in 5% CO 2 at 37 • C using a modified Trager and Jensen method [26] in fresh O+ human red blood cells at 3% hematocrit in RPMI culture media containing NaHCO 3 (Gibco, UK) and GlutaMAX supplement. This was supplemented with hypoxanthine (Gibco, Waltham, MA, USA), 25 mM of HEPES (Gibco, Drewton, UK), 0.5% Albumax II (Gibco, Waltham, MA, USA), and 20 µg/mL of gentamicin (Gibco, China). When needed, parasites were synchronized at the ring stage with a sorbitol (5%) treatment and further cultivated for one complete cycle (48 h) prior to the drug activity assays. Compounds dissolved in dimethyl sulfoxide (DMSO) were diluted in RPMI 1640 and mixed with the parasite cultures (1.5% hematocrit and 1% parasitemia, respectively) in 96-well plates to achieve final drug concentration levels of 10 µM for primary screening assays, and 10-0.078 µM for Metabolites 2023, 13, 298 4 of 11 the dose-dependent response assays. The final DMSO concentration per 100 µL culture per well was 0.1%. Artemisinin and chloroquine at 1 µM were used as positive drug controls, while (0.1%) DMSO was used as a negative drug control. Following a 72 h incubation at 37 • C, parasite growth was assessed using a SYBR green I-based DNA quantification assay. Briefly, 80 µL of parasitized erythrocytes were transferred to a dark plate and 40 µL of an SYBR green I-containing lysis buffer (3×) was added to the plate. The plate was incubated in the dark for 30 min and its fluorescence was measured using a Fluoroskan Ascent multi-well plate reader with excitation and emission wavelengths at 485 and 538 nm, respectively. The experiments were performed in triplicate and each one was repeated at least once. The concentrations at which 50% inhibition of growth (IC 50 values) was obtained were determined using GraphPad Prism 8.0, by plotting the logarithmic sample concentration on the x-axis against the percentage of inhibition on the y-axis.
The 13 C NMR ( Figure S10) spectrum displayed the signals of 27 carbon atoms sorted using both DEPT 135 ( Figure S11) and HSQC ( Figure S13) into four methyl groups; four methylenes; nine methines, including three furan methines at δ C 110.8, 142.1, and 144.0; and then quaternary carbons, including three carbonyls at δ C 173.0, 176.5, and 208.5. All these data were similar to those of 1-O-deacetylkhayanolide E (2a) [40]; the only difference was the coupling constant between protons H-5 and H-6 (J = 1.9 Hz instead of J = 8.7 Hz) in 1-O-deacetylkhayanolide E. This information led to the conclusion of a cis orientation for H-5/H-6. The 1 H-1 H COSY ( Figure S14) and the HMBC ( Figure S12) correlations (Figure 2) supported the heptacyclic skeleton of the proposed structure, which was then confirmed using single crystal X-ray diffraction analysis. Compound 2 contained twelve asymmetric carbon atoms; the absolute configurations were obtained with a Flack parameter of −0.02(3) (Figure 3). The C-6 configuration was determined to be R, while it was reported to be S for the known khayanolide E and 1-O-deacetylkhayanolide E (2a) [40]. Thus, compound 2 was determined to be (6R)-1-O-deacetylkhayanolide E, a C-6 epimer of 2a.
Fraction E showed the most significant activity (MIC values ranging from 3.9 to 250 µg/mL), and the best potency was observed against S. aureus (ATCC 25923, MIC = 3.9 µg/mL). Compound 9, isolated from fraction E, showed good activity (MIC = 7.8 µg/mL) against S. aureus (ATCC 25923), and may be the active principle. Compounds 3 isolated from fraction D exhibited moderate activity against S. aureus (ATCC 25923) and K. pneumoniae clinical isolate with a MIC value of 62.5 µg/mL. The other compounds (1, 5, 6, 7) were not active on the selected strains. The lack of activity of 1 compared to 3 could be attributed to the 24-OAc group. The activity of compound 3 was in agreement with the result obtained by Biavatti et al. [43] with the melianodiol on S. aureus (ATCC 6538, MIC = 25 µg/mL). However, its activity on K. pneumoniae strains was not yet evaluated. In addition, the activity of compound 9 was in accordance with that of Masika et al. [44], which showed the antibacterial activity of epicatechin against S. aureus strain (MIC = 250 µg/mL). In general, the observed antibacterial activity of limonoids was very poor. However, the crude extracts very often exhibited antibacterial activity [15,45,46]. The significant activity of T. monadelpha crude ethanol extract may be due to the synergic effect of its constituents.

Antiplasmodial Activity
The antiplasmodial activity (Table 3) of the extract and some isolated compounds (1-3, 5, 6, 9) against Plasmodium falciparum 3D7 (chloroquine sensitive/sulfadoxine resistant) was evaluated. Grandifotane A (6) and khayanolide D (5) showed good antiplasmodial activity with an IC 50 of 1.37 µM and 1.68 µM, respectively, compared to the reference compounds chloroquine and artemisinin with IC 50 values of 0.020 and 0.015, respectively. The mexicanolide-type limonoid grandifotane A (6) is several times more active than some previously reported compounds, such as 2,6-dihydroxyfissinolide (IC 50 = 0.12 mM), fissinolide (IC 50 = 48 µM), and 6-acetylsweitenolide (IC 50 = 8.80-33.12 µg/mL) [47,48]. However, the antiplasmodial activity of polyoxyphragmalin or phragmalin had not yet been investigated [47,49]. Here, we have reported the first evaluation of the antiplasmodial activity of the phragmalin limonoids (5). Limonoids are highly oxygenated tetranortriterpenoids with different cyclization and substitution patterns. The activity exhibited by compounds 5 and 6 opens further questions in terms of the mechanism of action and the structureactivity relationship. In addition, this result indicated that the Trichillia genus, and possibly the Meliaceae family in general, may be a promising source of potential antiplasmodial secondary metabolites.

Conclusions
The present study was undertaken to investigate the chemical constituents of the stems bark of T. monadelpha which, to our knowledge, had not yet been studied for either their chemical composition or their biological activities. The chemical investigations undertaken on an ethanol extract of the stems bark of the plant led to the isolation and characterization of thirteen compounds, including two new limonoids, namely 24-acetoxy-21,25-dihydroxy-21,23-epoxytirucall-7-en-3-one (1) and (6R)-1-O-deacetylkhayanolide E (2). Extracts, sub-extracts, fractions, and some isolated compounds were assessed for their antibacterial and/or antiplasmodial activities on seven bacterial strains and on one plansmodium strain, respectively. Fraction E from the EtOAc sub-extract showed the best activity against Staphylococcus aureus ATCC 25923 (MIC of 3.9 µg/mL). Epicatechin (9) and melianodiol (3) showed significant and moderated activities against Staphylococcus aureus ATCC 25923 with the MIC of 7.8 µg/mL and 62.5 µg/mL, respectively, while grandifotane A (6) and khayanolide D (5) showed good antiplasmodial activities with IC 50 values of 1.36 µM and 1.68 µM, respectively. Based on the above results, epicatechin and melianodiol could be the main antibacterial constituent of the stem bark of Trichilia monadelpha. As far as furanic limonoids are concerned, they were determined to be responsible for the antiplasmodial activity. For future re-investigations of the plant, collecting more starting material, evaluating its activity on other Plasmodium falciparum strains, as well as its toxicity and cytotoxicity, would all prove interesting areas of study.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; the collection, analyses, or interpretation of data; the writing of the manuscript; nor the decision to publish the results.